AN EVALUATION OF ANTIMICROBIAL EFFICACY OF ACNANO AGAINST SOME ACNE CAUSING MICROORGANISMS

Transcription

1 Original Research Manuscript International Journal of Drug Development & Research Jan-March 2010 Vol. 2 Issue 1 ISSN Available online IJDDR AN EVALUATION OF ANTIMICROBIAL EFFICACY OF ACNANO AGAINST SOME ACNE Naithani Vijay*, Kumar Shailesh, Srivastava Sanjay Mohan, Pathak Naveen & Chaudhary Manu *(Senior Manager R &D), Intellectual Scientific Division, Venus Medicine Research Centre Hill Top Industrial Estate, Bhatoli Kalan, Baddi, H.P India Phone No: , ABSTRACT This study was undertaken to determine the antimicrobial susceptibility of Acnano (a polyherbal nanoemulsion) against acne causing microbes Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis along with some other dermal disease causing microbes such as Pseudomonas aeruginosa, Morganella morganii, Enterobacter cloacae, Escherichia coli, Citrobacter braakii, Klebsiella pneumoniae, Acinetobacter baumannii, Proteus vulgaris, yeast such as Saccharomyces cerevisiae and fungi Candida albicans and Aspergillus niger. The agar well diffusion method, approved by NCCLS with the modification, was used. Student t test was performed using One way Analysis of Variance (ANOVA). This study showed that Acnano possesses potent antimicrobial activity against bacteria as well as fungi. Key Words : Acnano, antibacterial susceptibility, P. acnes, S. aureus, S. epidermidis Introduction Acne is an inflammatory skin disease characterized by pimples on the face. It affects individuals of all races covers 85% of teenagers, 42.5% of men, and 50.9% of women between the ages of 20 and 30 years [1,2]. Spontaneous regression usually occurs after age 20, but some patients may continue suffering during adult life [3]. In 2001, the global market for prescription acne products was estimated to be two billion dollars and the non-prescription market was estimated at two to four times of that size [4]. Lot of products are being developed to combat acne such as topical retinoids, benzoyl peroxide, salicylic acid etc. but due to high prevalence of antibioticresistant strains of Propionibacterium acnes, topical antibiotics are no longer effective as monotherapy [5]. Keeping this antibiotic resistance in consideration, *For correspondence E- mail: vijaynaithani@venusremedies.com Venus Medicine Research Centre, Baddi (India) has developed an anti-acne herbal nano-emulsion which is a perfect blend of essential oils and lemon effective against mild, moderate as well as chronic acne as well as other skin diseases including fungal diseases. It is an aromatic, clear, transparent light golden yellow viscous nano-emulsion which contains Melaleuca alternifolia oil, Rosmarinus officinalis oil, Mentha arvensis oil along with Citrus limon. This study was undertaken to determine the antimicrobial susceptibility of Acnano against acne causing microbes i.e., Propionibacterium acnes, Staphylococcus aureus & S. epidermidis and other microorganisms in comparison with standard Adapalene (0.1%). Material and Methods Acnano (Batch No. RND09H01, Mfg August 2009) was procured from Venus Medicine Int.J.Drug Dev. & Res. Jan-March 2010, 2(1):

2 Research Centre, Baddi (India). It was kept at room temperature away from direct light & moisture. Adapalene was purchased from Enaltec Labs Pvt. Ltd., Navi Mumbai , India. Selection of microorganism : All the microorganisms (Staphylococcus aureus, S. epidermidis & Propionibacterium acnes, Pseudomonas aeruginosa, Morganella morganii, Enterobacter cloacae, Escherichia coli, Citrobacter braakii, Klebsiella pneumoniae, Acinetobacter baumannii, Proteus vulgaris, A. niger, C. albicans and S. cerevisiae) used in the present study were Microbial Type Culture Collection (MTCC No. as per tables 2, 3 and 4), purchased from Institute of Microbial Technology, Sector 39-A, Chandigarh , India. Media and reagents:- Muller Hinton agar, Nutrient Broth, Sabouraud dextrose agar, Blood agar base, Barium chloride and Sulphuric acid. Preparation of Reagents/Buffers/Standards Muller Hinton agar, Sabouraud dextrose agar, Blood agar base medium and Nutrient broth were prepared as per manufacturer's (Hi Media Laboratories) instructions. Sterilized by autoclaving at 15 lbs pressure and 121 C for 15 minutes. 1. Preparation of Mueller Hinton Agar, Sabouraud Dextrose Agar and Blood agar Plates:- Immediately after autoclaving, it was allowed to cool in a 45 to 50 C water bath. Aseptically add 5% v/v sterile defibrinated blood to blood agar base medium. Freshly prepared and cooled medium were poured into a flat-bottomed petri dishes on a level, horizontal surface to give a uniform depth of approximately 4mm corresponding to 15 to 20 ml of medium for each plate with diameter of 90 mm. The agar medium was allowed to cool to room temperature. A representative sample of each batch of plates examined for sterility by incubating at 35 C for 24 hrs. 2. Inoculation Preparation:- At least three to five colonies of the same morphological type are selected from overnight plates cultures on non selective agar medium. The top of each colony is touched with a loop and the growth is transferred into a tube containing 4-5 ml of sterile Nutrient broth medium to produce a suspension which match the turbidity standard of 0.5 McFarland standard. 3. Preparation of McFarland Standard:- It is prepared by adding 0.5ml of M BaCl 2 (1.172 % w/v BaCl 2.H 2 O ) to 99.5 ml of 0.18 M H 2 SO 4 (1% v/v) with constant stirring. Using matches cuvettes with 1 cm. path length and water a blank standard the absorbance in a spectrophotometer at wave length of 625nm. the acceptable range of standard is Inoculation of Test Plates :- Optimally, within 15 minutes after adjusting the turbidity of the inoculum suspension, a sterile cotton swab is dipped into the adjusted suspension. The swab rotated several times and pressed firmly on the inside wall of the tube above the fluid level. This removes excess inoculum form the swab. The dried surface of a MH agar plates is inoculated with bacterial culture suspension except P. acne by streaking the swab over the entire sterile agar surface. The blood agar plates is inoculated with P. acne culture suspension and sabouraud dextrose agar plates is inoculated with fungus and yeast culture suspension by streaking the swab over the entire sterile agar surface. This procedure is repeated by streaking two more times, rotating the plate approx. 60 each time to ensure an ever Int.J.Drug Dev. & Res., Jan-March 2010, 2(1):

3 distribution of inoculum. As a final step, the rim of the agar is swabbed. Allowed the plates to dry until there is no visible surface moisture. 5. Boring in Petri-plates :- Created two bores in petri plates by using borer which has 6mm diameter and sterilized by dry heat sterilization (DHS). Individually, 100 micro liter of each Acnano and Adapalene (0.1%) were added in two bores each in each plate. All bacterial plates and their replicates were incubated at 35 O C for 24 hours whereas fungal plates were incubated at 25 O C for 96 hours. The bacterial plates of P. acnes were incubated anaerobically at 35 O C for 24 hours. Zone of inhibition was measured by antibiotic zone reader. Results and Discussion Already known active constituents of individual ingredients of Acnano have been given in Table 1. Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis are the major causal organism of acne [6,7,8]. Propionibacterium acnes is a relatively slow growing, typically aero-tolerant anaerobic gram positive bacterium that is linked to the skin condition acne. When a pore is blocked the bacterium overgrows and secretes chemicals that break down the wall of the pore, spilling bacteria such as Staphylococcus aureus onto the skin, and forming an acne lesion (folliculitis). P. acnes is an inhabitant of normal skin flora. It is implicated in the development of inflammatory acne by its ability to metabolize sebaceous triglycerides into fatty acids, which chemotactically attract neutrophils [9]. Zone of Inhibition (mm) of Acnano against these microorganisms was found to be better than the standard Adapalene (0.1%) Zone of Inhibition (mm) of Acnano against Propionibacterium acnes was found to be two times more than that of Adapalene (Table 2, Fig.1). Microbial efficacy of S. epidermidis & S. aureus were also better than the standard (Fig.2&3). Student t test was performed using One way Analysis of Variance (ANOVA). P value of Staphylococcus aureus, S. epidermidis & Propionibacterium acnes. was found to be highly significant p< Table1: Ingredients of Acnano and their anti-microbial active components Ingredient Melaleuca alternifolia oil (Tea tree oil) Active constituents Terpinen-4-ol, ã-terpinene, á-terpinene, 1,8-cineole etc. Rosmarinus officinalis oil (Rosemary oil) p-cymene, linalool, ã-terpinene, â-pinene, á-pinene, eucalyptol etc. Mentha arvensis oil (mint oil) Citrus limon Menthol Citric acid and ascorbic acid Table 2: Antibacterial Susceptibility Test (AST) of Acnano against Staphylococcus aureus, S. epidermidis & Propionibacterium acnes. Name of micro-organism MTCC No. Staphylococcus aureus 737 Zone diameter in mm Adapalene as standard (0.1%) Acnano 15.26± ±0.14 Staphylococcus epidermidis ± ±0.15 Propionibacterium acnes ± ±0.10 Int.J.Drug Dev. & Res., Jan-March 2010, 2(1):

4 CAUSING MICROORGANISMS Table 3: Antibacterial Susceptibility Test (AST) of Acnano against Pseudomonas aeruginosa, Morganella morganii, Enterobacter cloacae, Escherichia coli, Citrobacter braakii, Klebsiella pneumoniae, Acinetobacter baumannii & Proteus vulgaris. Name of micro-organism MTCC No. Zone diameter in mm Adapalene as standard (0.1%) Acnano Pseudomonas aeruginosa ± ±0.24 Morganella morganii 662 N il 12.41±0.11 Enterobacter cloacae ± ±0.23 Escherichia coli ± ±0.16 Citrobacter braakii ± ±0.18 Klebsiella pneumoniae ± ±0.09 Acinetobacter baumannii ± ±0.12 Proteus vulgaris 426 Nil 12.41±0.14 Table 4: Antibacterial Susceptibility Test (AST) of Acnano against A. niger, C. albicans & S. cerevisiae Name of micro-organism MTCC No. Aspergillus niger 1344 Zone diameter in mm Adapalene as standard (0.1%) Acnano 5.99± ±0.25 Candida albicans 227 Nil 20.78±0.31 Saccharomyces cerevisiae ± ±0.29 Int.J.Drug Dev. & Res., Jan-March 2010, 2(1):

5 CAUSING MICROORGANISMS Figure 1 : AST of Acnano against P.acnes (MTCC No ) Figure 2 : AST of Acnano against S.aureus (MTCC No ) Figure 3 : AST of Acnano against S.epidermidis (MTCC No ) Figure 4 : AST of Acnano against S.cerevisiae (MTCC No ) Figure 5 : AST of Acnano against A.niger (MTCC No ) Figure 6 : AST of Acnano against C.albicans (MTCC No ) Int.J.Drug Dev. & Res., Jan-March 2010, 2(1):

6 Besides main causal organism of acne, some other organisms such as Pseudomonas aeruginosa, Morganella morganii, Enterobacter cloacae, Escherichia coli, Citrobacter braakii, Klebsiella pneumoniae, Acinetobacter baumannii & Proteus vulgaris have also been associated with skin disorders [10,11,12,13,14,15,16, 17]. Efficacy of Acnano against these organisms was found to be better than that of Adapalene (0.1%) in all the microorganisms studied (Table 3). Antifungal effects of Acnano were also seen. Aspergillus niger, Candida albicans and Saccharomyces cerevisiae are the most common fungi that causes skin diseases [18, 19, 20, 21,22]. To verify that Acnano, besides being anti-acne product, can cure other skin related diseases also, antibacterial susceptiblity of Acnano against these fungi and yeast, was seen. Zone of inhibition of Acnano against Aspergillus niger, was found to be 4 times more than that of Adapalene (Fig.5). Efficacy of Acnano against Saccharomyces cerevisiae was found to be almost twice of Adapalene (Fig.4). Efficacy of Adapalene against Candida albicans was found to be nil but was well exhibited by Acnano (Table 4, Fig.6). All these results show that Acnano is potent against bacteria, fungi and yeast under study with particular mention of causal organisms of Acne. References 1) Alexis AF, Lamb A. Concomitant therapy for acne in patients with skin of color: a case-based approach. Dermatol Nurs 2009; 21(1): ) Lolis MS, Bowe WP, Shalita AR. Acne and systemic disease. Med Clin North Am 2009; 93(6): ) Ramos-e-Silva M, Carneiro SC. Acne vulgaris: review and guidelines. Dermatol Nurs 2009; 21(2): ) Bowe WP, Shalita AR. Effective over-the-counter acne treatments. Semin Cutan Med Surg 2008; 27:170. 5) Ghali F, Kang S, Leyden J, Shalita AR, Thiboutot DM. Changing the face of acne therapy. Cutis 2009; 83(2 Suppl): ) Nakatsuji T, Kao MC, Fang JY, Zouboulis CC, Zhang L, Gallo RL, Huang CM. Antimicrobial property of lauric acid against Propionibacterium acnes: its therapeutic potential for inflammatory acne vulgaris. J Invest Dermatol 2009; 129(10): ) Pechère M, Germanier L, Siegenthaler G, Pechère JC, Saurat JH. The antibacterial activity of topical retinoids: the case of retinaldehyde. Dermatology 2002; 205(2): ) Yamaguchi N, Satoh-Yamaguchi K, Ono M. In vitro evaluation of antibacterial, anticollagenase, and antioxidant activities of hop components (Humulus lupulus) addressing acne vulgaris. Phytomedicine 2009; 16(4): ) Swanson JK. Antibiotic Resistance of Propionibacterium acnes in Acne Vulgaris. Dermatology Nursing 2003; 15(4) : 10) Bachmeyer C, Landgraf N, Cordier F, Lemaitre P, Blum L. Acinetobacter baumanii folliculitis in a patient with AIDS. Clin Exp Dermatol 2005; 30(3): ) Boni R, Nehrhoff B. Treatment of gram-negative folliculitis in patients with acne. Am J Clin Dermatol 2003; 4(4): ) Gupta R, Rauf SJ, Singh S, Smith J, Agraharkar ML. Sepsis in a renal transplant recipient due to Citrobacter braakii. South Med J 2003; 96(8) : ) Isaac-Márquez AP, Lezama-Dávila CM. Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer. Reluctant response to antimonial treatment. Mem Inst Oswaldo Cruz 2003; 98(8): ) Kwakman PH, Van den Akker JP, Güçlü A, Aslami H, Binnekade JM, de Boer L, Boszhard L, Paulus F, Middelhoek P, te Velde AA, Vandenbroucke- Grauls CM, Schultz MJ, Zaat SA. Medical-grade honey kills antibiotic-resistant bacteria in vitro and eradicates skin colonization. Clin Infect Dis 2008; 46(11): Int.J.Drug Dev. & Res., Jan-March 2010, 2(1):

7 15) Leyden JJ, McGinley KJ, Mills OH. Pseudomonas aeruginosa gram-negative folliculitis. Arch Dermatol 1979; 115(10) : ) Papadopoulos CJ, Carson CF, Hammer KA, Riley TV. Susceptibility of pseudomonads to Melaleuca alternifolia (tea tree) oil and components. J Antimicrob Chemother 2006; 58(2): ) Samonis G, Karageorgopoulos DE, Kofteridis DP, Matthaiou DK, Sidiropoulou V, Maraki S, Falagas ME. Citrobacter infections in a general hospital: characteristics and outcomes. Eur J Clin Microbiol Infect Dis 2009; 28(1): ) Hammer KA, Carson CF, Riley TV. Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. J Antimicrob Chemother 2004; 53(6): ) Kishore N, Mishra AK, Chansouria JP. Fungitoxicity of essential oils against dermatophytes. Mycoses 1993; 36(5-6): ) Mohapatra S, Xess I, Swetha JV, Tanveer N, Asati D, Ramam M, Singh MK. Primary cutaneous aspergillosis due to Aspergillus niger in an immunocompetent patient. Indian J Med Microbiol 2009; 27(4) : ) Williams JS, Mufti GJ, Powell S, Salisbury JR, Higgins EM. Saccharomyces cerevisiae emboli in an immuno-compromised patient with relapsed acute myeloid leukaemia. Clin Exp Dermatol 2007; 32(4): ) Yehia MA, El-Ammawi TS, Al-Mazidi KM, Abu El- Ela MA, Al-Ajmi HS. The Spectrum of Fungal Infections with a Special Reference to Dermatophytoses in the Capital Area of Kuwait During : A Retrospective Analysis. Mycopathologia 2009 Author Information: Dr. Vijay Naithani is working as Senior Manager R &D, Intellectual Scientific Division, Venus Medicine Research Centre, Baddi, H.P Article History: Date of Submission: Date of Acceptance: Conflict of Interest: NIL Source of support: NONE ****************************************** Int.J.Drug Dev. & Res., Jan-March 2010, 2(1):